1 Course title: Hematology (1) Course code: MLHE-201 Supervisor: Prof Course title: Hematology (1) Course code: MLHE-201 Supervisor: Prof. Dr Magda SultanOutcome :The student will know :-The types of hemolytic anemias-The diagnosis of hemolytic anemias-The types of hereditary hemolytic anemias.-The diagnosis of hereditary hemolytic anemias-Types of acquired hemolytic anemias-The diagnosis of acquired hemolytic anemias-The laboratory tests needed for diagnosis
2 Hemolytic anemia Hemolytic anemia = decreased levels of red blood cells (anemia) because of their destruction (hemolysis)A red blood cell survives 120 daysThe spleen is the main organ which removes old RBCs from the blood.
3 Causes of hemolytic anemias can be either: 1 - hereditary.2 - acquired.
4 Hereditary Hemolytic anemia Defects of hemoglobinThalassaemia, Sickle cell anemiaDefects of the red cell membraneHereditary spherocytosis, Hereditary elliptocytosisDefective red cell metabolism (enzymes)G6PD deficiency. P K deficiency
6 Signs of hemolytic anemia: Physical Symptoms of anemiaJaundicePallorSplenomegaly / hepatosplenomegaly
7 Laboratory features Anemia of increased destruction Normochromic, normocytic anemiaShort RBC survivalReticulocytes increasedIncreased indirect bilirubinIncreased LDH*Peripheral blood smear microscopy:fragments of the red blood cells and spherocytesNormoblasts can be present.Bone marrow smear microscopy:Erytrhroid hyperplasia
8 Hereditary Hemolytic anemia Defects of hemoglobinThalassaemia, Sickle cell anemiaDefects of the red cell membraneHereditary spherocytosis, Hereditary elliptocytosisDefective red cell metabolismG6PD deficiency. P K deficiency
9 Sickle cell anaemiaThe abnormalities of the gene may result from substitution of single amino acid (Substitution of glutamic acid by valine )The Hb is stable when oxygenated state and become unstable and polymerized on deoxygenated state
10 Sickle cell anaemiaPolymerization will lead to precipitation of Hb. The cell become deformed (sickle shape) and very sticky leading to vascular occlusion and small infarction to the affected areas.Short life span of cells leading to chronic anaemia,
11 2- Hemoglobin electrophoresis: Increased hemoglobin S Sickle cell anaemiaDiagnosis1-Sickling test2- Hemoglobin electrophoresis:Increased hemoglobin S(90% Hgb S, 10% Hgb F, small fraction of Hgb A2)
12 HEMOGLOBINNORMAL ADULT RBC CONSISTS OF 3 FORMS OF Hb: HbA - 2 α and 2 β globin chains HbA2 – 2 α and 2 δ globin chains HbF - 2 α and 2 γ globin chains
15 ThalassaemiaMeans decrease synthesis of one ofthe globin chain which form normal hemoglobin.(HbA - 2 α and 2 β globin chainsHbA2 – 2 α and 2 δ globin chainsHbF - 2 α and 2 γ globin chains ). The defect may be in alpha chain ( thalassaemia),Beta chain ( thalassaemia) or Delta chain ( thalassaemia)
16 Beta Thalassaemia Anaemia skeletal changes & hyper metabolism Defective chain synthesisExcess chain Precipitationcell membrane damageCirculating Red cellBone marrowAnaemiaHemolyticIneffective erythropoiesisErythropoietin increasedblood transfusion Iron absorptionBone marrow expansionIron overloadskeletal changes & hyper metabolismComplication and death
17 Beta-Thalassemia major laboratory features Severe anemiaBlood film: microcytic hypochromic , target cells, basophylic stippling, reticulocytes increased and normoblasts .Marrow: marked erythroid hyperplasia, Shortened red cell survivalHaemoglobin electrophoresis :Fetal hemoglobin > 90%, HbA absent, HbA2 low/normal/high
18 HEREDITARY SPHEROCYTOSIS Defective or absent spectrin moleculeLeads to loss of RBC membrane, leading to spherocytosisDecreased deformability of cellIncreased osmotic fragilityExtravascular hemolysis in spleen
19 Hereditary spherocytosis (HS) Laboratory features hemolytic anemia blood smear spherocytes increased osmotic fragility time
21 Glucose 6-Phosphate Dehydrogenase Functions Regenerates NADPH, allowing regeneration of glutathioneProtects against oxidative stressLack of G6PD leads to hemolysis during oxidative stressInfectionMedicationsFava beansOxidative stress leads to Heinz body formation, extravascular hemolysis
29 Procedure of DATTake 2-3 drops of blood to be tested in a clean labeled tube.Wash the red cells 3-4 times in a large volume of saline to remove free globulin molecules. Remove all supernatant after each wash. Completely decant the final supernatant wash.Add 2 drops of polyspecific AHG serum in 1 drop of sensitized washed red cells or in 1 drop of 3-5 % suspension of sensitized cells immediately.Mix, Centrifuge at 1000 rpm for 1 minutes immediately.Gently shake the tube to dislodge the cell button and see for agglutination, use optical aid if needed. Record the result.Add 1 drop of IgG coated red cells to a negative test. Mix, centrifuge at 1000 rpm for 1 min. Immediately look for agglutination. If a negative result (no agglutination) is obtained the test result is invalid and whole test should be repeated. If agglutination is obtained, the result is valid.
31 Procedure:Place 2-3 drops of the test serum in a tube. Serum should be fresh for detecting complement components and complement binding antibodies, otherwise, fresh AB serum should be added to it.Add 1 drop of 3-5% suspension of washed O Rh (D) positive red cells to the serum in the tube.Mix and incubate at 37°C for minutes.Centrifuge at 1000 rpm for 1 minutes.Examine for hemolysis and/or agglutination. Use optical aid if necessary. Agglutination at this stage indicates the presence of saline (complete) antibodies.If no agglutination is seen, wash cells 3-4 times in large volume of saline. Decant supernatant in each wash as completely as possible.
32 Procedure: Add 2 drops of AHG serum to the cells. Mix and centrifuge at 1000 rpm for 1 minutes immediately.Gently shake the tube to dislodge the button and examine for agglutination, using optical aid. Record the result.Add 1 drop of IgG coated red cells to any test that is negative. Mix and centrifuge at 1000 rpm for 1 minutes. Look for agglutination. If there is no agglutination, the test result is invalid and the whole test is repeated. If agglutination is obtained the result is valid.Auto control should be kept with IAT.